The long term goal of this project is to develop, validate, and commercialize an assay to improve screening of C9ORF72, a gene on chromosome 9 that is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Expansions of a guanine and cytosine rich hexanucleotide repeat (GGGGCC) in the non-coding region of C9ORF72 is associated with up to 67 percent of familial ALS, making it the most prevalent genetic mutation for a disease with an annual economic burden of $433M in the US. The expansion also appears in about 25 percent of familial FTD, as well as 7 percent of sporadic ALS and 5 percent of sporadic FTD. The C9ORF72 region is difficult to size accurately because most expansions in affected individuals are more than 700 repeats in length. Currently testing relies on homebrew PCR-based assays for accurate sizing of <35 repeats, or, separately, Southern blot analysis for crude sizing of >35 repeats. A solution is needed to enable high throughput, reliable, sensitive, and accurate sizing of the repeat region for both short (<35 repeats) and long expansions (>35 to 1000 repeats) in a single assay. The proposed assay offers a solution to these technical challenges based on the repeat-primed assay platform (Amplidex(R) FMR1 PCR) that Asuragen has developed and successfully commercialized for fragile X syndrome, a CGG triplet repeat disorder. We will leverage >5 years of experience in optimizing high performance diagnostic assays for GC- rich repeat sequences, including methods for the routine amplification of at least 1300 CGG repeats, to develop an accurate and robust single tube molecular diagnostic test for C9ORF72. The specific aims of this proposal are: Aim 1. Optimize the detection of C9ORF72 hexanucleotide repeat expansions using a repeat-primed PCR strategy. Aim 2. Optimize and validate a single tube repeat sizing assay using previously characterized genomic DNA samples from Coriell cell repositories. The development of an improved, information-rich assay for C9ORF72 will be useful as a screening and diagnostic test for ALS and FTD as well as a clinical research tool to identify intermediate and/or expanded repeat sizes that are potentially relevant to other forms of age-onset neurodegeneration, to further an understanding of known and novel genotype-phenotype associations, and to enable opportunities for targeted therapeutics and clinical trials.